(a) Field of the Invention
The present invention relates to the use of a novel bacteria of genus Erwinia, referred to as Erwinia taxi, isolated from Taxus canadensis, for the production of paclitaxel and related taxanes. There is disclosed the methods of isolation of this bacteria and the screening tests that were used to prove the production of paclitaxel by said bacteria.
(b) Description of Prior Art
Paclitaxel, also referred to as Taxol.TM., has been first identified in 1971 by Wani and collaborators (Wani MC et al., 1971, J. Am. Chem. Soc., 93:2325-2327) following a screening program of plant extracts of the National Cancer Institute. This complex diterpene demonstrated cytotoxic activity against several cancer cell lines such as KB cells, mouse leukemia cells (Wani MC et al., 1971, J. Am. Chem. Soc., 93:2325-2327), and against in vivo systems such as intraperitoneally implanted B16 murine melanoma and MX-1 mammary tumor xenografted in mice (National Cancer Institute: NCI Annual Report to the Food and Drug Administration: Taxol (IND 22850, NSC 125973), Bethesda, Md.: NCI 1989). Later, activity has also been reported against different types of cancer; platinum-refractory ovarian cancer (McGuire WP et al., 1989, Ann. Intl. Med., 11:273-279), refractory ovarian cancer (Holmes FA et al., 1991, J. Natl. Cancer Inst., 83:1797-1805), breast cancer, lung cancer (Murphy WK et al., 1992, Phase II study of Taxol (NCS 125973) in patients with non-small-cell lung cancer (NSCLC), Proc. ASCO II (abstrc.):294), head and neck cancer (Forastiere AA et al., Sep. 23-24, 1992, Phase II trial on Taxol in head and neck cancer: An Eastern Cooperative Oncology Group Study, Proceedings of the Second National Cancer Institute Workshop on Taxol and Taxus (abstrc.), Alexandria, Va.). Several phase III clinical trials began in 1992 and paclitaxel has been approved in December 1992 by the Food and Drug Administration. It is now being used in the treatment of some cancers, mainly ovarian cancer.
Paclitaxel differs from other cytotoxic drugs by its unique mechanism of action. It interferes with cell division by manipulating the molecular regulation of the cell cycle. Paclitaxel binds to tubulin, the major structural component of microtubules that are present in all eukaryotic cells. Those microtubules form the mitotic spindle in the G2/M phase of cell division. Unlike other antimitotic agents such as vinca alkaloids and colchicine, which inhibit the polymerization of tubulin, paclitaxel promotes this assembly of tubulin and stabilizes the resulting microtubules. Thus, the division of the cell in two equal daughter cells is interrupted. Microtubules also regulate cell shape, they anchor surface receptors in the plasma membrane, they are involved in motility and in the formation of channels in neurotransmitter secretion. Within the cell, a dynamic equilibrium exists between microtubules and their depolymerized tubulin dimers. Paclitaxel disrupts this equilibrium thus preventing the transition from Go/G1 through S phase which causes a lethal metaphase arrest.
Paclitaxel is presently commercialized by Bristol-Myers Squibb (BMS) and the supply of the drug is problematic. The substance is extracted from the bark of the slow-growing Pacific yew, Taxus brevifolia. Actually, 16 000 pounds of bark are necessary to produce 1 kg of paclitaxel. The low yield of the isolation of paclitaxel (0,016 g %) and the limited availability of the trees have forced the scientific community to find alternative sources of producing paclitaxel.
Among those alternative sources, total synthesis of the drug would be an interesting compromise. Total synthesis of paclitaxel has been achieved in 1994 by Nicolaou et al. (1994, Nature, 367:630-634). The process is somehow multi-stepped and the overall yield has made this approach economically unrealizable. Nevertheless, semisynthesis of paclitaxel from its natural precursor, 10-deacetylbaccatin III, is now possible. This precursor can be extracted from a renewable source: the needles and twigs of the European yew Taxus baccata.
Plant cell culture of taxus species is another approach explored by many scientists. This process is limited by the quantity of paclitaxel that can be produced and the length of incubation time required to yield paclitaxel levels comparable with those produced by the intact plant. Those reasons have made this method economically unrealizable.
In U.S. Pat. No. 5,322,779, Gary A. Strobel et al. disclosed a fungus isolated from the bark of a sample of Taxus brevifolia which is able to synthesize paclitaxel at a level of only 24-50 ng/L after 3 weeks of cultivation. The utilization of this fungus with a slow growth at an industrial level would require large volumes of culture and long periods of incubation in order to extract significant amounts of the drug, which reduce the profitability of the process. With a microorganism having a higher doubling time and a higher rate of metabolism, larger quantities of paclitaxel could be extracted in a short delay. Bacteria possess these characteristics of growth, specially fermentative microorganisms.
Plants are hosts of a variety of microbes including fungus and bacteria. It is known that some of these microbes are able to synthesize secondary plant compounds. So, some genetic material could be exchanged between the host and microorganisms. It would be highly suitable if a bacteria could be used for the production of paclitaxel. As mentioned earlier, bacteria have, in general, a high doubling time and can be easily grown. Therefore, it would become a non-expensive renewable source of paclitaxel that can be applied to the pharmaceutical industry.
It would be highly desirable to be provided with a bacteria for the mass production of paclitaxel and related taxanes.